The detection of specified antigens (defined as a substance whose introduction into an animal stimulates the production of antibodies capable of reacting specifically therewith), haptens (a substance requiring additional accessory materials before its introduction into an animal stimulates the production of antibodies specific therefor), and the like substances (hereinafter collectively referred to as ligands) in body fluids such as blood, sputum, urine and the like has in recent years become of utmost importance in both the research and clinical environments. The detection of ligands, particularly antigens or antibodies capable of specifically combining therewith (hereinafter collectively termed "anti-ligands" or "ligand binding partner" which terms are also meant to include antibody fragments such as F(ab), F(ab)' etc.) can often be related to various disease states and consequently is extremely useful in diagnosis, in gaining basic understandings concerning the genesis of disease, and in monitoring the effectiveness of therapies therefor. Many schemes for detecting ligands or anit-ligands have evolved over recent years based on the selective, immunological reactivity which characterizes these substances. Generally, these schemes are collectively termed immunoassays.
This invention is particularly concerned with the class of immunoassays which measure changes in fluorescence polarization and depolarization (hereinafter simply referred to as fluorescence depolarization or fluorescence polarization since each refers to the same process but from different viewpoints) for the detection of ligands. In particular, the fluorescence depolarization methods have been most popularly utilized in connection with drug monitoring.
To date, however, fluorescence depolarization has been substantially limited to monitoring antigens or ligands of small molecular weight. Such low molecular weight ligands, on the order of less than 1000 daltons, rotate rapidly in solution. Accordingly, when a small fluorescent molecule is attached to the light antigen or ligand, it also rotates rapidly. Thus, when the fluorescent molecule is excited by polarized light, the resultant fluorescent light radiated by the fluorescent molecule becomes partially depolarized due to the rapid rotation of the fluorescent molecule-ligand. The speed of rotation, and concommitantly the amount of depolarization, dramatically decreases when the low molecular weight ligand becomes substantially heavier such as when it becomes bound to a comparatively much larger antibody (i.e., anti-ligand) molecule. Similarly, limited depolarization is observed when the fluorescent molecule itself is bound to a high molecular weight molecule (e.g., over 1000 daltons). The amount of depolarization as a function of molecular weight (determined by whether an anti-ligand binds to a ligand) can thus serve as the basis for an immunoassay for low molecular weight ligands.
In such a fluorescence depolarization immunoassay, the observation of a decrease in depolarization (e.g., the maintenance of polarization) indicates increased binding of anti-ligand to the fluorescently labeled ligand since such a binding results in a larger molecule which rotates slowly and is thus a less effective depolarizing agent. If, on the other hand, the sample contains ligands which compete with the fluorescently labeled ligands for binding sites on the anti-ligand, then fewer anti-ligand molecules are available to bind to the fluorescently labeled ligands and an increasing level of depolarization is observed. As may be readily appreciated, quantitation of such an assay may be conveniently accomplished using standard preparations for comparison with samples containing unknown levels of the low molecular weight ligand. In fact, this technique is currently being employed by Abbott in their commercially available TDX instrument such as is described in U.S. Pat. No. 4,269,511 and U.S. Pat. No. 4,420,568.
The latter patent, to Wang et al., describes fluorescent depolarization immunoassays utilizing substituted Triazinylaminofluoresceins. A review of this patent, however, highlights the limitations of the fluorescent depolarization techniques to ligands of low molecular weight, generally in the range of 50-4000 and most preferably those within a range of 100-2000. Many investigators have heretofore, however, regarded the practical upper limit as being somewhat lower than those of Wang, more on the order of approximately 1000. As previously described, these limits exist because as the ligands become significantly larger, they no longer rotate rapidly. Consequently, the attached fluorescent molecule also does not rotate rapidly. As a result, little or no depolarization can be observed even before an anti-ligand binds the large molecular weight ligand. In addition, the large molecular weight ligands are no longer significantly affected from a percentage change in weight viewpoint by the binding of an anti-ligand, and thus, they show little increased depolarization when subsequently bound to an anti-ligand. Consequently, the sensitivity of the polarization assay for a ligand rapidly drops off with increasing molecular weight of the ligand.
It is an object of the present invention to remove these limitations by providing methods for detecting the presence of large molecular weight ligands while still employing the principles of fluorescence polarization.
It is yet another object of the present invention to provide methods suitable for all types of ligands regardless of weight, not just the low molecular weight protein free haptens which the Wang et al. U.S. Pat. No. 4,420,568 describes.